Ovarian cancer is the most common cause of cancer death from gynecologic tumors in the United States. While some ovarian tumors initially respond to first line chemotherapy, the majority of advanced-stage ovarian cancers develop chemoresistance and patients succumb to their disease. Without a full molecular understanding of how resistance is mediated, even promising new therapies could ultimately fail. Thus, we propose to define these mechanisms and develop tools to anticipate when resistance to chemotherapy will occur. This knowledge will be critical to guide therapy choice and will provide insight into new therapies targeting the mechanisms of resistance. Our innovative idea was to identify mechanisms of chemoresistance in ovarian cancer by an unbiased screen. Indeed, our survival based genome-wide shRNA screen enabled us to identify genes, whose loss is essential for the cancer cells to resist cisplatin therapy. Significantly, several of these genes ae mutated or lost in ovarian cancer and associated with patient response to therapy indicating that our screening strategy was a success. Now, with these genes in hand, our proposed research plan in Aim 1 is to further validate genes in vitro and in vivo. In Aim 2, we will define how depletion of these genes confers resistance. Not only will we learn how loss of these genes unleashes resistance in ovarian cancer, but also these studies will help identify what resistance pathways should be inhibited to enhance the efficacy of therapy. In particular, we will test the hypothesis that genes whose loss confers cisplatin resistance also confer a drug tolerant state (DTS), in which chromatin modifications and DNA damage responses are reduced. Thus, we will determine if knocking down validated gene candidates induces a DTS, or, conversely, ectopic expression of validated gene candidates reverses a DTS. Because epigenetic drugs that preserve chromatin modifications and restore a robust DNA damage response sensitize cells in a DTS, we will test whether epigenetic drugs will re-sensitize cisplatin resistant ovarian cancer cells. Finally, in Aim 3, we will test the hypothesis that genes identified in our screen will be useful as biomarkers. Given that resistance uniquely requires that these genes be depleted, low expression of these genes in ovarian tumors could predict poor response to cisplatin. Thus, it will be critical not only to determine whether these genes are tumor suppressors disrupted in ovarian cancer, but also whether these genes provide a signature predicting cisplatin response in patients, ideas we will test with outstanding reagents and expertise provided by our clinical collaborators. Altogether, the research plan proposed here will provide opportunities to define, detect (biomarkers), and ultimately disable compensatory mechanisms in ovarian cancer.